Inc/GFP chimera protein-based interactomics reveals host cellular interactions of Cps0558, a novel Chlamydia psittaci inclusion protein

Abstract The obligate intracellular Gram-negative bacterium Chlamydia psittaci, a zoonotic pathogen transmissible between birds and humans, has played a pioneering role in research on its membrane-bound replicative niche termed the inclusion. Inclusion membrane proteins (Inc proteins) are crucial for Chlamydia–host interactions and were first identified in C. psittaci. This study investigates putative C. psittaci Inc proteins by a combination of in silico analyses, immunofluorescence and, strikingly, a new Inc/GFP chimera protein-based interactomics approach to identify host cellular interaction partners. Here, we report a novel C. psittaci Inc protein, Cps0558, along with respective host cellular interaction partners, in particular ACAD11, which is involved in lipid metabolism. We confirm their physical interaction in the native infection context, supporting the physiological relevance of our chimera-based screen. Furthermore, new interaction partners for the known Inc protein IncA are identified, revealing a potential role of IncA as modulator of the host ubiquitylation system. These results provide further insights into the biology of C. psittaci and present a novel tool for studying Inc proteins under conditions closely resembling their natural tertiary structure.